Selective binding materials

ABSTRACT

A template (a molecule preferably of molecular size &gt;500 Da, or a larger entity such as a cell, virus or tissue sample) is dissolved or suspended in a fluid. The fluid is frozen, and the template is removed (e.g. by dissolution or electrophoresis, or mechanically) to leave an “imprinted” frozen fluid. This is capable of selectively adsorbing the template substance. It is usable as a separation medium, a recognition element in sensors and assays, and as a catalyst.

TECHNICAL FIELD

[0001] The present invention relates to selective binding materials,their preparation and uses.

BACKGROUND ART

[0002] Polyakov in the 1930's described substrate-specific materialsprepared by condensing silicic acid in the presence of templatemolecules (Zhur.Fiz.Khim.2:799 (1931); 10:100(1937); 4:454 (1933)). Morerecent workers have used organic monomers, polymerized in the presenceof template molecules (e.g. U.S. Pat. Nos. 5,110,833, 5,728,296,5756717, WO 9641173).

DISCLOSURE OF INVENTION

[0003] In a first aspect the present invention provides a process forpreparing a selective binding material capable of selectively binding atemplate material comprising:

[0004] (a) preparing a composition comprising template material and acarrier fluid;

[0005] (b) freezing the composition; and

[0006] (c) at least partially removing template material from the frozencomposition so as to leave frozen carrier fluid with binding sites wheretemplate material has been removed.

[0007] The template material may be selected from proteins, biologicalreceptors, nucleic acids, chromosomes, cells, viruses, microorganisms,tissue samples, carbohydrates, oligosaccharides, polysaccharides,nucleoproteins, mucoproteins, lipoproteins, synthetic proteins,glycoproteins, glucosaminoglycans, steroids, immunosuppressants,hormones, heparin, antibiotics, vitamins and drugs.

[0008] The carrier fluid may be selected from water, aqueous solvents,aqueous solutions, inorganic liquids, organic solvents, metals, fusibleorganic compounds, fusible inorganic compounds, polymers and gases. Whenthe template material is molecular, the fluid will generally be asolvent therefor. The composition may be a suspension, as it generallywill be if the template material is a complex entity, such as a tissuesample. Molten metals will generally be restricted to low melting metalse.g. mercury and gallium. Gases that can be liquefied and solidified bycontrol of temperature and/or pressure may also be used.

[0009] Preferred compositions are aqueous solutions, e.g. of proteins.

[0010] A composition may be a 2-phase system, e.g. with the templatematerial partitioned between immiscible solvents (e.g. water and anorganic solvent such as chloroform or ether). Only the higher-freezingone of these may be frozen in step (b).

[0011] When the template material is soluble, step (c) will usuallyinvolve its dissolution, preferably using a solvent in which the frozencarrier fluid is not substantially soluble (e.g. using chloroform,acetonitrile or other organic solvents or mixtures to remove materialsfrom ice). Suitable materials may be removed by electrodialysis.Particularly for particulate material, mechanical removal may beappropriate.

[0012] The invention preferably uses template molecules of molecularsize exceeding 500 Da.

[0013] The frozen imprinted fluid has predetermined affinity andspecificity superior to non-imprinted material and can be prepared muchmore easily than traditional cross-linked imprinted polymers. Materialsprepared as described in this invention can be used as adsorbents inseparation and purification, as catalysts and as recognition materialsin sensors and assays.

[0014] In a second aspect the present invention provides a selectivebinding material as prepared by the above process.

[0015] In further aspects the invention provides uses of such materialsexploiting their selective binding abilities. Use as a separation matrixis exemplified below. An example of use in a sensor or array couldinvolve using our materials in place of antibodies in immunosensors andassays employing them. An example of use as a catalyst would involveemploying a transition state analogue as the template material toproduce a material capable of catalysing a reaction that proceedsthrough such a transition state.

[0016] In a yet further aspect the invention provides a process forpreparing and using a selective binding material comprising:

[0017] (a) preparing a composition comprising template material and acarrier fluid;

[0018] (b) freezing the composition; and

[0019] (c) contacting the frozen composition with a solution containinga molecular species which selectively interacts with template materialin the frozen composition.

[0020] Traditional imprinting polymerisation includes formation of arigid polymer network around a template which may be a small organicsubstance, such as a drug, or a protein or even a cell. Molecularlyimprinted polymers (MIPs) prepared in this way consist of monomer unitslinked together by predominantly covalent interactions.

[0021] The present invention, in various aspects, describes theformation of the substrate-specific material by freezing the solvent orother fluid in the presence of a template. In contrast to traditionalMIP preparation, where solvent plays a secondary role, being at most areaction medium which facilitates the complex formation between thefunctional monomers and the template, this new approach relies on theability of the frozen solvent to act as adsorbent. When prepared in thepresence of template, frozen solvent contains cavities occupied bytemplate molecules which can be emptied and used for rebinding of thetemplate molecules. The structure of the imprints formed should becomplementary to the structure of the template or molecules withstructure and shape similar to those of the template molecules.

[0022] In embodiments in which template material is not removed from thefrozen carrier fluid, template will remain immobilised. At least part ofthe molecules, suspension particles or tissue will remain exposed andavailable for a subsequent action (recognition, binding, catalysis,sensing etc.). Such material can be used as a separation matrix, elementof sensor or assay and as a catalyst.

BRIEF DESCRIPTION OF DRAWINGS

[0023]FIG. 1 shows adsorption isotherms comparing adsorption of atemplate material to ice formed in the presence of the material (“mip”)and ice formed in its absence.

[0024] FIGS. 2(a) and 2(b) show capacity factors K′ (a) and separationfactors (b) of a range of analytes passed through a column containing aselective binding material prepared in the presence of one of them.

[0025]FIG. 3 is a trace showing the result of electrophoresis of amixture of D- and L-phenylalanine in a capillary filled with ice, formedin the presence of L-phenylalanine.

MODES FOR CARRYING OUT THE INVENTION

[0026] 1. Preparation of Isoproterenol-Specific Adsorbent.

[0027] Sephadex G25, 30 mg/sample were transferred into the wells offiltration microplates and swelled overnight in 0.2 ml 1 mg/ml solutionof (+)-isoproterenol (+)-bitartrate salt. The excess of solution wasremoved by filtration under reduced pressure and adsorbent was flushedtwice with 0.2 ml chloroform. The swelled adsorbent was frozen for 12hours at −15° C. Adsorbent was washed four times with acetonitrile at−15° C. Blank material was prepared in the same way but in the absenceof isoproterenol. The adsorption properties of prepared adsorbents wereevaluated by adding 0.15 ml aliquot of the corresponding analyte in theconcentration ranging from 0.1 to 10 mg/ml. Concentration ofisoproterenol in solution was measured spectrophotometrically at 280 nm.Results of the sorption analysis indicate that ice, prepared in thepresence of template has higher affinity to the template than blanksample FIG. 1). At the same time Sephadex G25 itself has no affinity toisoproterenol.

[0028] The same material, prepared in the presence of isoproterenol hasbeen analysed by HPLC in experiments with compounds having structuresimilar to that of isoproterenol. Results of this analysis indicate thatthe ice has highest affinity to the target compound presented insolution (FIG. 2). An HPLC column (100×4.6 mm i.d.) was packed withapproximately 1 g Sephadex G25 swelled in 1 mg/ml (+) isoproterenol(MIP) or water (Blank). Column was washed with chloroform and frozen at−15° C. for 12 hours. All chromatographic experiments were performed at−10° C. using detection at 280 nm and flow rate 1 ml/min. The column waswashed on-line with acetonitrile until a stable baseline was obtainedand solvent replaced with 10% aqueous acetonitrile. 20 μl solution ofthe analyte in 0.1 mg/ml concentration were used for the injections. Ipr(+)-(+) isoproterenol, Ipr (−)-(−) isoproterenol, Phe—phenylephrine,Pro—propranolol, Epi—epinephrine, Nep—norepinephrine.

[0029] 2. Preparation of Material Specific for L-Phenylalanine.

[0030] The experiment was done using capillary electrophoresis unitQuanta 4000E, Waters. A 35 cm portion of capillary (fused silica, 80 cm,100 um internal diameter—Polymicro Technologies, Hallow, UK) wasinserted into insulated tubes connected with the refrigerating unit andcooled to a temperature of −14.5° C. The capillary was filled with a 2mM solution of L-Phe in 9.5 mM HCl, and incubated at −14.5° C. for 1hour, to allow complete refrigeration of the liquid inside the capillarylumen. To elute the template from the ice a voltage of 13 kV was appliedto the capillary and maintained until the baseline is stabilised. Thesample, D-Phe, L-Phe or the racemate, diluted to a concentration of 2 mMin 9.5 mM HCl were injected hydrodinamically into the capillary for 6sec. The run was performed by applying 13 kV (the current is 10 uA). Thesame experiment was performed using blank ice, formed in the absence ofthe template. The result of this analysis shown that the ice formed inthe capillary in the presence of the L-Phe has enhanced affinity to thetemplate and can be used to separate optical enantiomers (FIG. 4). Noseparation of L-Phe and D-Phe was observed when control (blank) icefilled capillary was used.

1. A process for preparing a selective binding material capable ofselectively binding a template material comprising: (a) preparing acomposition comprising template material and a carrier fluid; (b)freezing the composition; and (c) at least partially removing templatematerial from the frozen composition so as to leave frozen carrier fluidwith binding sites where template material has been removed.
 2. Aprocess according to claim 1 wherein in step (c) template material isremoved mechanically.
 3. A process according to claim 1 wherein in step(c) template material is removed by washing with a solvent therefor. 4.A process according to claim 1 wherein in step (c) template material isremoved by electrodialysis.
 5. A process for preparing and using aselective binding material comprising: (a) preparing a compositioncomprising template material and a carrier fluid; (b) freezing thecomposition; and (c) contacting the frozen composition with a solutioncontaining a molecular species which selectively interacts with templatematerial in the frozen composition.
 6. A process according to claim 5wherein said step (c) is used to effect molecular recognition, selectivebinding, sensing, assaying or catalysis.
 7. A process according to anypreceding claim where the template material is selected from proteins,biological receptors, nucleic acids, chromosomes, cells, viruses,microorganisms, tissue samples, carbohydrates, oligosaccharides,polysaccharides, nucleoproteins, mucoproteins, lipoproteins, syntheticproteins, glycoproteins, glucosaminoglycans, steroids,immunosuppressants, hormones, heparin, antibiotics, vitamins and drugs.8. A process according to any preceding claim wherein the carrier fluidis selected from water, aqueous solvents, inorganic liquids, organicsolvents, metals, fusible organic compounds, fusible inorganiccompounds, polymers and gases.
 9. A process according to any precedingclaim wherein said carrier fluid is a liquid and said compositionprepared in step (a) is a solution of the template material.
 10. Aprocess according to claim 9 where the solution is in water and thetemplate material is a protein.
 11. A process according to any of claims1-8 where the template material is immobilised onto a solid support. 12.A process according to any of claims 1-11 where the template material isdistributed between two phases, which have different melting points. 13.A selective binding material as prepared by the process of any of claims1-4 or 6-12 consisting of a frozen carrier fluid having binding siteswhence template material has been removed.
 14. Use of the material ofclaim 13 as a separation matrix.
 15. Use of the material of claim 12 asa recognition element for a sensor or assay for sensing or assaying thetemplate material or an analogue thereof.
 16. Use of the material ofclaim 12 as a catalyst.